Originally published September, 1998
Part 1: Research fundamentals and industry catalystsCliff Henke
Is the adoption of DNA-chip technologies a nanopipe dream, or the next medical revolution? In this three-part series, IVD Technology explores the potential of this emerging technology for diagnostics. Part 2 is also available for on-line viewing.
As is often the case with breakthrough technologies, a great deal of hyperbole has surrounded the development of micro- and nanoarray diagnostic technologies, popularly known as DNA or genetic chips. Yet much of the optimism expressed in such statements is almost certainly justified. Industry analysts agree that when their promise begins to be fulfilledprobably sometime early in the next decadeDNA chips will usher in a new era in medical care.
Researchers in the field expect that DNA chips will enable cliniciansand in some cases even patients themselvesto quickly and inexpensively detect the presence of a whole array of genetically based diseases and conditions, including AIDS, Alzheimer's disease, cystic fibrosis, and some forms of cancer. Moreover, the technology could make it possible to conduct widespread disease screening cost-effectively, and to monitor the effectiveness of patient therapies more effectively. In the meantime, however, considerable work remains to be done. So far, only a few companies have commercialized DNA-chip products, and the barriers to market entry remain great.
This article, the first of a three-part series on DNA-chip technologies, will review the theoretical underpinnings of the technologies and examine the market forces that are driving product development. The second installment will look at the state of the art, including the various competing technologies in this embryonic field. The final article will examine the obstacles to commercialization that companies in this new marketplace will have to overcome, as well as prospective near- and long-term applications for DNA-chip technologies.
Although the DNA-chip marketplace is in its infancy, with considerable challenges remaining to be overcome, the speed with which manufacturers are progressing toward commercialization will soon make DNA chips viable alternatives to traditional chemical assays. Indeed, if analysts are correct, within a decade they will usher in a new era in diagnosis and treatment for diseases and conditions that have genetic origins.
A variety of recent technological breakthroughs have made possible the development of DNA chips. Fundamentally, however, genetic chips are the result of achievements in two fields: molecular biology and microfabrication technology.
Molecular Biology. Especially as it has been catalyzed by the work of the Human Genome Project (HGP), research in molecular biology has laid the groundwork for the development of clinical laboratory tests and therapies involving genetic probes. Fundamental advances include the use of polymerase chain reaction (PCR) or other amplification techniques to make copies of a nucleic acid sample, which can then be tested using a genetic probe, that is, a known gene and its molecular structure. Also essential to the development of DNA chips has been the creation of gene sequencers, machines that have automated the biochemical tests necessary to identify genetic sequences using gene probes.
But the roots of gene probe technologies go back much further, to molecular biology discoveries made decades ago. Most important of these are the base-pairing rules discovered in the 1950s by James Watson and Francis Crick. Watson and Crick determined that the DNA molecules found in living organisms are composed of a structure of two twisted strands (the famous double helix) latticed together with pairs of nitrogenous bases: adenine, cytosine, guanine, and thymine. They also discovered that these bases always recur in the same two pairs: adenine with thymine, and cytosine with guanine. Thus, by knowing part of the molecular structure of a specific genetic segment, one can determine the other part.
Moreover, these uniquely complementary strands of DNA can be sought out by using one of the strands to test for its biochemical mate; this is the basis of a gene probe. The process of one strand of DNA matching up with its counterpart strand is called hybridization. It is this technique that is used to determine a base-pair sequence in a DNA sample, also called genetic sequencing.
Hybridization can be performed either in solution or on a solid support. In traditional gene sequencing, the most common format for hybridization is the Southern blot, which uses a nitrocellulose sheet. However, some companies use solution-based processes, and there is considerable experimentation in the field to develop new hybridization formats. DNA-chip manufacturers are also exploring variations of both solid-phase and solution-based hybridization for use in their microassays. At present, however, most DNA-chip companies use a solid-phase technique.
DNA chips are designed to identify hybridization products in the same fashion as with traditional sequencers. Once hybridization has been completed, phosphorescent chemicals that bind to the hybridized sequences are scanned with a light source, making it easy to detect their presence with automated colorimetric or fluorimetric equipment.
Microfabrication Technologies. The second technological trend that is making DNA-chip products possible encompasses the steady improvements in nano- and microscale fabrication techniques. Developed initially for use in computer chip manufacturing, these techniques are now being exploited in a variety of other disciplines, including DNA-chip manufacturing. These achievements have made possible the application of organic structures (e.g., segments of DNA and reagents) onto a substrate of inorganic materials. Unlike computer chips, which use silicon-based wafers, DNA microassays are fabricated onto glass or plastic wafers or are placed in tiny glass tubes and reservoirs.
Although the fundamental principles of molecular biology apply to the design of all DNA chips currently under development or commercially available, approaches to the fabrication of substrates for such products vary considerably. While some developers use manufacturing techniques very similar to those used in computer chip fabrication, others are exploring techniques very different from semiconductor manufacturing. At the computer chip end of that continuum is the approach taken by such companies as Affymetrix (Santa Clara, CA). To produce its Genechip line of products, Affymetrix bonds hundreds of genetic sequences onto the surface of a microchip using photolithographic processes such as photosensitive masks, chemical doping layers, and other techniques used in computer chip fabrication.
How Genetic Sequencing Works
Sequencing, the process of finding the molecular structure of a DNA fragment, employs the Watson-Crick rules of hybridization, whereby each strand of DNA can bond only to a chemical mirror image via two sets of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T).
Step 1: Determine chemical structure of fragment.
Representing all or part of a DNA strand of interest, short fragments of DNA (typically involving 525 base pairs) are identified.
Step 2: Separate strands.
DNA is denatured (separated) and placed in solution or on a solid substrate, forming a reference segment for the DNA fragment of interest.
Step 3: Introduce sample.
Unknown DNA sample is introduced to the reference segment. If present, the complement of the reference segment will hybridize (bond) to it.
Step 4: Identify result.
Chemicals that bond to successful hybridization help researchers identify results. Such chemicals are typically photosensitive (fluorescent or chemiluminescent), which helps researchers confirm results.
Probe arrays are manufactured by Affymetrix's proprietary, light-directed chemical synthesis process, which combines solid-phase chemical synthesis with photolithographic fabrication techniques employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density arrays of oligonucleotides, with each probe occupying a predefined position in the array. Multiple probe arrays are synthesized simultaneously on a large glass wafer. This parallel process enhances reproducibility and helps achieve economies of scale. The wafers are then diced, and individual probe arrays are packaged in injection-molded plastic cartridges, which protect them from the environment and serve as chambers for hybridization.
Another manufacturing approach involves the deposition of gene probes onto the chip substrate using a tiny droplet sprayer that resembles an ink-jet printer. This approach is being used by Combion (Redwood City, CA), Rosetta (Seattle), ProtoGene Laboratories (Palo Alto, CA), and Affymetrix. (Two of these firms illustrate the heavy involvement of higher education in this emerging field; Combion was created from research conducted at the California Institute of Technology, while Rosetta uses techniques developed at the University of Washington.) Manufacturers spray a chemical solution containing the gene probes in a pattern onto the chip substrate, in the same fashion as in other clinical lab tests.
Some companies, such as Nanogen (San Diego), use robots to deposit the gene probes onto the substrate. Nanogen uses electrophoresis to speed up hybridization.
Yet another approach is the use of gels in a solution-based process. Scientists at the Argonne National Laboratory (Argonne, IL) hope to find commercial backing for this approach within the next two years.
Thus, the concept behind DNA chips is simply that of miniaturizing the gene sequencing technologies already being developed, so that many assays and their related procedures can be performed together. DNA chips will give researchers the ability to analyze thousands of genes at once, and may also make it possible to conduct very elaborate diagnostic procedures in such small settings as a physician's office or even with mobile equipment used at the point of care.
Product Development Catalysts
Enormous forces are impelling the development of DNA chips. The most important of these is the federal government's Human Genome Project. Begun in 1990, the HGP is a federally funded and directed research endeavor involving scientists throughout the world. Its goal is to locate and characterize every gene on every human chromosome by 2003. Thanks to technologies such as DNA chips, researchers are now hinting that the project may be completed by the end of this decade, three years early.
The HGP has contributed in two significant ways to the development of microassays. First, it has created a genomics market. Genomics is the science of discovering, locating, and characterizing genes in organisms. HGP grants issued by the National Institutes of Health and other institutions all over the world have increased the appetite for genetic chips as part of the HGP's race to completion. As basic research dollars of the HGP have led to more information about the human genome, pharmaceutical companies and venture capital funds have poured in further billions of dollars to commercialize applications of genomic discoveries.
How DNA Chips Are Made
Using conventional techniques such as polymerase chain reaction and biochemical synthesis, strands of identified DNA are made and purified. A variety of probes are available from commercial sources, many of which also offer custom production services.
Step 2: Manufacture substrate wafer.
Companies use photolithography and other nanomanufacturing techniques to turn glass and plastic wafers into receptacles for the DNA probes.
Step 3: Deposit genetic sequences.
Manufacturers use a variety of processes ranging from electrophoretic bonding to robotic deposition to adhere genetic material to the substrate. Cleanroom conditions and standards must be observed to attain the degree of contamination control needed during the deposition process.
Step 4: Customer use.
Completed chips are checked for quality, packaged, labeled, and sent to clients. In use, chips enable researchers to identify the components of probes deposited on the chips, usually with the help of phosphorescent tags.
In turn, HGP discoveries have helped researchers develop more and better tools for their gene-hunting workthe second manner in which the project has contributed to the development of DNA chips. Several companies have either designed and manufactured chips to help tackle specific questions for research establishments engaged in the HGP, or have done so to help pharmaceutical and diagnostics manufacturers in their commercial research projects. Moreover, the additional information acquired in both government-funded and private-sector research has led to product ideas with commercial potential outside medicine. Toxicologists, for instance, have sought to adapt DNA-chip technologies for use in agricultural research and in conducting environmental impact studies.
For some companies, the key by-product of HGP research is directly related to the field of medical diagnostics. Nanogen, for example, will use its platform technology created initially for genomics research as a base for developing commercial applications in the infectious disease diagnostic market. According to Kieran Gallahue, executive vice president of Nanogen, the company is currently looking into partnering with some other large diagnostic companies. However, he adds, the company will not be ready to market any products until late 1999.
Another large group of contributors to DNA-chip development has been made up of pharmaceutical companies. Most microarray manufacturers already have partnerships and long-term commercial arrangements with the world's major drug companies to develop products that meet pharmaceutical research needs. For example, several such teams are working on ways to more quickly identify therapies that are effective in identifying and preventing cancer-causing mutations in the p53 gene.
Other government programs have also helped develop genetic chips. For example, Affymetrix and Hyseq (Sunnyvale, CA) each received $2 million in funding from the Department of Commerce's Advanced Technology Project, which awards matching grants to firms whose promising research is not sufficiently developed to attract commercial or investor support but might show great economic promise in the long run.
The Department of Energy's national laboratories have been another catalyst of research and development in this field. Argonne National Laboratory's work, mentioned earlier, is one example of this. The Sandia National Laboratories have also helped some companies with research in microlithography processes that can be used in DNA-chip fabrication.
As DNA-chip companies prepare to bring their products to market, major technological and regulatory challenges still lie ahead of them. The technology trade-offs will involve finding ways to increase the number of arrays on a single chip, as well as increasing the rate of production to meet expected demand. Currently, few manufacturers are producing chips beyond a pilot-scale production rate.
A second technology challenge involves achieving all these market-oriented parameters at a cost that supports a commercially acceptable price. Currently, DNA chips cost between $100 and $450 each. In a tight managed-care marketplace that places a premium on technologies that can either show quick savings or more-efficient results, some analysts say that such unit prices will limit the growth of the DNA-chip market.
As products are readied for the marketplace, they will also encounter regulatory challenges, such as ensuring that manufacturing processes meet current quality systems and CLIA standards. These regulatory and technical manufacturing hurdles will be discussed further in subsequent articles scheduled in this series.
Although the DNA chips now commercially available are less than a few years old, there is already a growing commercial interest in these devices for research, diagnostic, and therapeutic applications. Enormous hurdles must still be overcome, as will be discussed in the next installment of this series. However, the pace of activity is so great that observers are already confident that widespread application of DNA chips is less than a decade away.
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Cliff Henke is a freelance writer based in Southern California.
Illustrations by Robert Margulies.
Continue to Part 2 of this series.